Members of the genus Rickettsia are the etiologic agents of epidemic and endemic typhus and rocky mountain and other spotted fevers. These diseases pose significant health threats worldwide. Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm ofa eukaryotic host cell. R prowazekii is able to exploit this intracellular niche in animals as diverse as arthropods and humans, allowing it to be vectored by the human body louse. This proposal is focused on the characterization of the protein expression patterns ofR. prowazekii. Important targets for a proteome approach are the bacterial pathogens of humans, organisms that exhibit unique biologies, and unfortunately, in the current human environment, potential bioterrorist agents. R. prowazekii fits all three of these criteria and in addition, due to its small proteome, can serve as a model for complete proteome analysis. The specific focus of this proposal is on the development and application ofproteomic techniques to address questions about the pathogen R. prowazkeii. In Specific Aim 1 our goal is to develop and apply high- throughput, global liquid chromatography-mass spectrometry technology to obtain the complete proteome of the extensively studied R. prowazeldi Madrid E strain propagated under standard conditions. We will apply this technology in Specific Aim 2 to identify global patterns of rickettsial protein expression. Rickettsial proteins expressed under different conditions that reflect the unique biology of this pathogen will be examined. These will include strain differences, host cell differences, temperature variation, and changes resulting l_om rickettsial growth. In Specific Aim 3 we will address a critical extension of host cell variation by examining the proteome ofR. prowazkeii growing in an arthropod vector. This interface is a critical component of rickettsial survival. Identification of differences in rickettsial protein expression will increase our understanding of this dynamic process as well as identify potentially new targets for vaccines and diagnostic reagents.